Cyclic oligoadenylate signalling mediatesMycobacterium tuberculosisCRISPR defence

Abstract

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE is achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show that the mechanism can be reprogrammed to operate as a cyclic tetra-adenylate (cA4) system by replacing the effector protein. These observations demonstrate that M. tuberculosis has a fullyfunctioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

“…Tsac 2833 mediated plasmid immunity from a reprogrammed type III system in E. coliPlasmids pCsm1-5_ DCsm6 (containing the type III Csm interference genes cas10, csm3, csm4, csm5 from M. tuberculosis and csm2 from M. canettii), pCRISPR_TetR (containing M. tuberculosis cas6 and tetracycline resistance gene-targeting CRISPR array), pRAT-Target (tetracyclineresistance, target plasmid) and M. tuberculosis (Mtb)Csm6/ Thioalkalivibrio sulfidiphilus (Tsu)Csx1 expression constructs have been described previously 10 . pRAT-Duet was constructed by replacing the pUC19 lacZa gene of pRAT-Target with the MCSs of pACYCDuet-1 by restriction digest (5'-NcoI, 3'-XhoI).…”

“…Each nuclease was cloned with and without the viral ring nuclease; pRAT-Duet without insert and pRAT-Duet containing only the viral ring nuclease were used as controls. The plasmid transformation assay was carried out essentially as described in 10 . E. coli C43 containing pCsm1-5_DCsm6 and pCRISPR_TetR were transformed by heat shock with 100 ng of pRAT-Duet target plasmid containing different combinations of cOA-dependent nuclease and viral ring nuclease.…”

“…cOA molecules are synthesised with a range of ring sizes with 3-6 AMP subunits (denoted cA3, cA4 etc.) by the cyclase domain of the Cas10 protein [1][2][3]9,10 . cOA binds to a specific protein domain, known as a CARF (CRISPR Associated Rossman Fold) domain.…”

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23

“…We used a portion of the tetracycline resistance gene as the target sequence and selected for tetracycline resistance after transformation of the cells with the target/effector plasmid. For active effectors, we expected to see fewer transformants compared to inactive effectors due to target depletion and/or programmed cell death (Figure 2A) [33].…”

“…Effector proteins were cloned into pRATDuet [33]. This plasmid carries the pRSF1030 replicon from RSFDuet™ (Novagen, Merck Millipore), araC and the araBAD promoter from pBAD/His (Invitrogen), the tetracycline resistance gene from pACE2 (Geneva Biotech, Genève, CH) and the two MCSs from pACYCDuet™-1 (Novagen, Merck Millipore).…”